Chromatographic techniques are well known in the art as means for separating components (solutes) present in a mixture. These techniques are particularly useful in the chemical and biotechnological arts. True chromatography describes the separation of solutes according to their different partitioning between two (or three) phases. The phases generally are solid and liquid, and solute partitioning results in their differing mobilities through a layer of solid, typically particulate, matrix in the presence of a flowing phase. Solute transfer through the layer may be along a pressure gradient, generally referred to as xe2x80x9cliquid chromatographyxe2x80x9d. Typically, the sample to be separated is applied to a column filled with pellets or grains of a chromatography separation medium, and a solvent flow is maintained through the column at a steady rate. Components of the mixture are carried along by the solvent flow until each substance exits the column as a xe2x80x9cpeakxe2x80x9d in the output, different peaks being more or less broad and overlapping.
Chromatographic matrices can separate components by any of a number of criteria, including size, electrical charge, hydrophobic interaction, and/or specific affinity for the matrix or binding sites thereon. Because the components in the mixture will vary in their affinity for the matrix, their partitioning as they pass through the matrix separates the components so that they exit the matrix sequentially, separated temporally and spatially. Determination of the location of the various separated components, or of a given component of interest within the sequence, generally is achieved by collecting the fluid phase exiting the matrix (i.e., the effluent stream) as a series of fractions and sampling these fractions to identify their contents by any of a number of means known in the art.
Resolution of the various components in the mixture depends on several considerations, chief among them being the partitioning ability of the matrix and the system""s theoretical plate height and plate number (see infra). In general, a large surface area-to-volume ratio is desired. Matrices for liquid chromatography systems typically are housed in cylindrical chromatography systems known as columns. In electrophoresis systems, high resolution also demands efficient removal of the heat generated by the applied electric field. Capillary electrophoresis, or other electrophoretic modules which provide a large surface area-to-volume ratio dissipate Joule heat well, allowing rapid analysis without significant loss of resolution.
Techniques are known for treating the chromatography medium to enhance the affinity of the column generally for cationic or anionic substances, or to cause a reversible bonding to particular chemical groups or biologically active materials, so that samples containing these groups or materials may be releasably bound to the column and subsequently eluted.
To achieve a particular separation, the general practice of chromatographic separation involves identifying or selecting a particular medium or coated medium, and an optimum solvent, solvent flow rate, pH, ionic concentrations and other environmental conditions, such that the starting mixture will separate into a number of relatively narrow bands and such that at least the substance of interest passes, or may be made to pass, as a distinct output.
The determination of an appropriate set of separation conditions for a particular substance, which may have an as yet undetermined chemical structure and conformation and unknown chromatographic affinities, is a task that involves experience, experimentation, intuition and luck. Because of the complex dependence of the transport and adsorption mechanisms of biomolecules on multiple different conditions, further experimentation is usually necessary even when it is desired only to scale up a known process to operate at greater speed or to utilize a larger column. In order to meet the separation objectives of high purity, high speed and/or high volume separation, a very large number of separation conditions must be experimentally analyzed to determine one suitable set of operating conditions.
In general, the transport of material in a separation column proceeds on a macroscopic level by flow past and between the grains or pellets of the chromatography medium, while the degree of separation and column capacity are governed more by the rates at which the particular components diffuse along branching paths into and out of pores in the medium, and are repeatedly adsorbed and released along the diffusion path. By increasing the flow rate to increase process output, one generally broadens the eluted peak width of each component, thus sacrificing the resolution and hence the purity of the separated components; above a certain flow rate threshold, premature solute breakthrough may occur.
The need to monitor a product""s status during its synthesis or purification is well known in the art. Status monitoring is particularly important in multistep preparation protocols. Frequently, the identity and, often, the quality of a product in a mixture must be determined at each step. Product monitoring also may be used as part of a feedback system to adjust process parameters. Generally, identification is determined using a previously established criterion for identification, for example, a characteristic absorbance measured at a given wavelength. If the product of interest is a protein, identification also may be by molecular weight, activity, and/or immunoaffinity.
It is an object of the invention to provide a rapid, adaptable, and repeatable system and apparatus for identifying the presence and/or location of a molecule of interest during any preparative or analytic protocol. The ultimate goal is to separate one or more components of a protein mixture by exploiting the benefits of high speed chromatographic techniques. Objects of the invention include two dimensional analysis to enhance resolving power of a chromatographic system, real time monitoring of solute concentration in a process mixture, detection of trace solute contaminants in a solution that contains a major amount of a dissolved product, rapid determination of the presence and location of a solute in a chromatography effluent during, e.g., any step of a preparative procedure, production of a profile of a mixture representative of the nature and relative concentration of structured variants of a given solute, and the rapid assessment of the success of a purification or separation protocol.
The invention features an apparatus and methods for the rapid and efficient separation of proteins and other biological macromolecules. The apparatus includes sample input means, a first liquid chromatography column, a multiport injection valve connecting the sample input means to the column, a second chromatography column in communication with the multiport injection valve, the second column being operative successively with or alternatively to the first column, pump means for providing variable pressure delivery of a solution to the column via the multiport valve, and program means for specifying a sequence of system control programs.
In preferred embodiments, the apparatus further includes control means in communication with the pump means for controlling the pressure of delivery of the solution; and solution input means including plural solution reservoirs, and a mixing valve, connecting the solution input means to the sample input means, operative to mix solution from the reservoir, wherein the program means specifies the mixing of solution by the mixing valve, and the delivery of the mixed solution to the column via the multiport injection valve; and detector means for detecting and recording column output; and matching means for identifying a pattern of detected output data, the template matching means being operatively keyed to means for developing a control program for liquid chromatography separation.
In another embodiment, the invention features an apparatus for the separation of proteins, which includes first and second liquid chromatography columns, means for introducing a solution into a first said column, multiple multi-port valves in communication with the first and second columns through which solution is transported, and output means comprising a detector and data collector.
Preferably, this embodiment includes pump means for introducing solution into the first column; control means in communication with the pump means for controlling the pressure of delivery of the solvent. Preferably, the multiple valves include first, second, and third valves, and the solution is introduced through the first and second valves into the first column, through the second and third valves into the second column, and through the third valve into the output means. The first valve also includes multiple ports which communicate with each other in an adjacent clockwise or counterclockwise direction, and a loop connecting two non-adjacent ports. The solution introducing means comprises sample input means which includes a sample reservoir. The sample input means may further include a sample pump, and the solution introducing means may include plural solution reservoirs, a valve for selection and mixing solutions, and a pump for delivering solution to the first column. The output means may further include a fourth multi-port valve connecting the detector to the data collector. The detector may be a UV detector. The output means may further include a pH/conductivity detector in communication with the UV detector and the data collector through the fourth multi-port valve.
Preferably, the column has a first and a second end and at least one of the first or second columns is packed with a chromatography matrix which confers on the packed column a transit time from the first to the second end of less than five minutes. The chromatography matrix itself may be perfusive.
In another embodiment of the invention, the apparatus includes a multiport mixing valve for mixing sample with one or more buffers to produce a sample mix, plural liquid chromatography columns, each column includes a first and a second end, a multiport injection valve in communication with the sample mixing valve and the first end of each of the chromatography columns, an output system including at least one of an output signal recording system and an output sample collection system, wherein the output system is in communication with the second end of each of the columns, and controls means for operating the multiport injection valve to successively and alternately apply the mixed solution from the mixing valve to the first and to the second column in coordination with operation of the output system to run a sequence of separations for the preparation or analysis of a protein.
Preferably, the apparatus further includes a sample input system comprising plural solution reservoirs and a sample reservoir.
In another embodiment, the apparatus further includes plural chromatography columns, each column packed with a particulate matrix separation medium and having a characteristic transit time for proteins of under five minutes between a column input and a column output ends, sample input means including an input valve for delivery of solutions to one column at the column input end and a multiport valve for mixing solutions provided to the input valve, column output means for detecting column output including means for detection and providing a signal indicative thereof, and control means for operating the sample input means to perform a sequence of successive separations in one column by providing in successive separation cycles different mixes of fluids to the input valve. The control means may further include switching means for alternatively utilizing one of the chromatography columns while cleanng another, thus providing a substantially continuous operating sequence of outputs from successively utilized columns. The apparatus may further include program means for specifying a sequence of separation process control programs to be successively run during operation. The program means may specify a separation program in which first and second columns are utilized successively for separating proteins in the sample.
Preferably, one column of the apparatus includes an ion exchange chromatography matrix. Alternatively or additionally, one column may include a reverse phase chromatography matrix. The apparatus is preferably used for preparation and analysis of a sample, where the first column specifies a preparative parameter and the second column specifies an analytical parameter. The program may thus specify a substantially continuous preparation of a separated sample in the first column and intermittent analysis of the first column output via the second column. Each of the first and second columns, individually, may be removable and replaceable by third and fourth columns, respectively.
In another embodiment, the apparatus includes first and second multiport valves, each valve including a sample loop, for holding a defined sample volume, connecting two ports of each valve, a liquid chromatography column in communication with each valve, a sample feed line in communication with each valve, detector means in communication with the second valve for detecting output, and control means for operating the multiport valves to switch between a collection line comprising the sample feed line wherein plural sample volumes are introduced and a detection line including the chromatography column, wherein one sample volume is passed through the detector means and another is passed through the column and detector means.
In preferred embodiments, the collecting line further includes in successive order (a) the first sample loop connecting within the first valve a first port to a second port, (b) the sample feed line connecting the first valve second port with the second valve first port, and (c) the second sample loop connecting within the second valve the first port to the second port. The detection line may further include in successive order (a) the first sample loop connecting within the first valve a first port to a third port, (b) the chromatography column connecting the first valve third port with the second valve third port, (c) the second sample loop connecting within the second valve the third port to the second port, and (d) a shunt connecting the second valve second port with the detector. In other preferred embodiments, additional multiport valves may be present; for example, a third multiport valve positioned in order between the first and the second valves, and connecting the chromatography column to these valves.
In this embodiment of the invention, the apparatus is capable of holding two defined volumes of sample, a nonadsorbed sample which has bypassed the column and an adsorbed sample which has passed through the matrix and thus lacks most of the target solute. The adsorbed sample solution will exit the matrix in-line with the sample solution that bypassed the matrix and that is contained within the second sample loop. The two sample solutions will then flow through a detector and result in a graph with two well-defined peak That is, when the feed solution that bypassed the matrix reaches, the detector, a peak representative of the concentration of all solutes in the effluent, i.e., the target and nontarget solutes together, will result. This will be followed by a second peak representative of the concentration of non-target solutes only. The difference in peak areas divided by the area of the peaks representing total solutes in the sample is a measure of the purity of the sample.
Thus, all information necessary to calculate the target solute and/or impurities concentrations is available in a defined sample volume as soon as the sample has been passed through the column matrix. If desired, the target solute can be eluted from the matrix and its concentration can be determined independently of the concentrations of feed solution containing all solutes and the adsorbed solution containing only nontarget solutes.
Advantages of this embodiment of the invention include rapid monitoring of the presence, quantity, and/or purity of a target solute in a product sample, during a preparative procedure. The rapidity of the analysis, e.g., it can be performed in as little as 10 seconds, reduces the analytical burden of monitoring a preparative procedure and the downtime necessary to determine a subsequent preparative step. Impurities that are monitored in the rapid monitoring system include proteins, nucleic acids, endotoxins, or any biological molecule detectable in the sample.
In another aspect, the invention features methods of analyzing proteins of a sample. The methods include the following embodiments. One embodiment is a method of analysis of proteins of a sample which includes introducing a sample to a first column comprising an input and an output end, wherein the first column separates components of the sample and produces a first effluent stream of separated components, interrupting the first effluent stream at a pre-determined position to collect a fraction of separated components, introducing the component portion to a second column, wherein the second column separates components of that fraction to produce a second effluent stream comprising separated components of the fraction, and detecting components of the second effluent stream.
Preferably, there is substantially continuous preparation of a separated sample in the first column and intermittent analysis of the first column output via the second column. One column may include an ion exchange chromatography matrix; and/or one column may include a reverse phase chromatography matrix. The first column effluent may be diverted back to the first column input end via a multi-port valve, and this effluent may contain a substantially pure product. The second column may be designed to provide a pattern of output data determinative of the number of times the first effluent is diverted back to the first column input end. Preferably, a portion of the effluent stream may be directed to the second column by switching a multi-port valve.
In another embodiment, this aspect of the invention features a method of analysis of proteins of a sample, and includes introducing a sample to a first column comprising an input and an output end, wherein the first column separates components of the sample and produces a first effluent stream of separated components, diverting the first effluent stream to a second column comprising input and output ends that selectively removes a target component of the sample from the stream to produce a second effluent stream comprising substantially all components of the first effluent stream except the target component.
Preferably, the method further includes detecting the components of the first and second effluent streams.
In another embodiment, the invention features a method of analysis of proteins of a sample, which includes introducing a sample to a first column, wherein the first column, which includes input and output ends, selectively removes a target component from the sample to produce a first effluent stream comprising substantially all components of the sample except the target component, diverting the first effluent stream to a second column, comprising input and output ends, that separates components of the sample and produces a second effluent stream of separated components.
Preferably, this method includes detecting the output of the first and second effluent streams.
In another embodiment, the invention features a method of analysis of proteins of a sample which includes introducing a sample comprising a target protein and trace solutes to a first column, including input and output ends and a target-specific adsorbing means, wherein the first column selectively retains a target protein from the sample to produce a first effluent stream substantially lacking the target protein, diverting the first effluent stream to a second column, including input and output ends and a trace solute adsorbing means, that selectively adsorbs trace solutes from the stream to produce a second effluent stream, eluting the trace solutes from the second column, and detecting the eluted trace solutes.
Preferably, this method also includes a first column target adsorbing means which includes a target-specific affinity chromatography matrix, which may include be a target protein-specific immunoglobulin. Preferably, the trace solute adsorbing means comprises a means for nonspecifically binding proteins.
Preferably, in the embodiments of this aspect of the invention, the transit time between input of said sample into the input end of the first column and the output end of the second column is less than 10 minutes; most preferably, less than 7 minutes. The first effluent stream is directed to the second column by switching a multi-port valve. Each column may be packed with a particle matrix separation medium which confers on each column a characteristic transit time for proteins of under five minutes between each column input and column output ends. Preferably, the matrix is a perfusive chromatography matrix.
The methods and apparatus of the invention are rapid, reliable, and adaptable, and are particularly useful in the preparation of biological macromolecules, particularly in the separation and purification of proteins. The chromatography system described herein has advantages when used as a two column or one column system to separate components of a given sample; e.g., information from a first run may be used to calibrate a second run. The first and second columns may be readily regenerated with recycling solvents, allowing the systems to be used repeatedly throughout a given procedure. In addition, other advantages include automated preparation or analysis of a sample, e.g., a combined preparative/analytical procedure in which a first column is used to separate a component of a sample, and the second column is used to interrupt the purification procedure at any chosen time to assess the purity of the sample, thus providing a two-dimensional chromatographic analysis; analysis of the purity of a sample by removing the purified product in a first column, and concentrating and eluting contaminants in the second column; and analysis of both the concentration and purity of a product, and analysis of the product itself, i.e., structural variants which constitute the product peak or the proportion in the sample of pure product and impurities.